Isolation and characterization of ornitho‐kininogen

Ornitho‐kininogen was purified from chicken blood plasma by a two‐stage method using chromatography on columns of S‐alkylated papain‐Cellulofine and DEAE‐5PW. The yield was 1.7 mg from 44 ml plasma. The isolated preparation gave a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS‐PAGE) with or without 2‐mercaptoethanol and on disc/polyacrylamide gel electrophoresis. The relative molecular mass, M_r, of ornitho‐kininogen was estimated as 74000 on SDS‐PAGE using the Ferguson plot method. Ornitho‐kininogen was found to have the similar properties to those of mammalian high‐M_r kininogen, in terms of the amino acid composition, molecular mass, and susceptibility to plasma kallikrein. No kininogen corresponding to mammalian low‐M_r kininogen and rat T‐kininogen could be detected in chicken plasma. In fact, ornitho‐kininogen was degraded rapidly by bovine plasma kallikrein, liberating a kinin. This kinin was isolated from the digest by reversed‐phase HPLC. The primary structure of the isolated kinin was determined as Arg¹‐Pro²‐Pro³‐Gly⁴‐Phe⁵‐Thr⁶‐Pro⁷‐Leu⁸‐Arg⁹. The sequence of this peptide, named ornitho‐kinin, was similar to that of bradykinin except for the substitution of Thr⁶ and Leu⁸ for Ser⁶ and Phe⁸. The isolated ornitho‐kinin induced a contraction of chicken smooth muscle and had a strong hypotensive effect in the chicken. However, it did not contract the isolated rat uterus. It is suggested that this specificity difference is due to the replacement of Phe⁸ by Leu⁸. The sequence of residues 1–30 of ornitho‐kininogen exhibited 43% identity with that of bovine kininogen.